Isolation and Characterization of Matrix Associated Region DNA Fragments in Rice (Oryza sativa L.)

To investigate the interactions between chromosomal DNA andnuclear matrices in higher plants, matrix associated regions(MARs) of rice (Oryza sativa L.) DNAs were cloned. First, weprepared nuclear matrices from isolated nuclei by digestingthem with EcoRl and then extracting with 2 M NaCl. About 6%of the total DNA remained in the nuclear matrices after thisdigestion and extraction. The residual DNA fragments in thenuclear matrices were cloned. Some of the cloned DNA fragmentsshowed binding to certain nuclear proteins. One of the MAR fragmentscontained sequences related to known consensus motifs and ahairpin loop structure. A method is presented for isolationof matrix associated region (MAR) DNAs from plant cells. (Received January 13, 1997; Accepted July 10, 1997)     

Quantitative immunofluorescence and immunoelectron microscopy of the topoisomerase IIα associated with nuclear matrices from wild-type and drug-resistant Chinese hamster ovary cell lines

Topo IIα is considered an important constituent of the nuclear matrix, serving as a fastener of DNA loops to the underlying filamentous scaffolding network. To further define a mechanism of drug resistance to topo II poisons, we studied the quantity of topo IIα associated with the nuclear matrix in drug-resistant SMR₁₆ and parental cells in the presence and absence of VP-16. Nuclear matrices were prepared from nuclei isolated in EDTA buffer, followed by nuclease digestion with DNase II in the absence of RNase treatment and extraction with 2 M NaCl. Whole-mount spreading of residual structures permits, by means of isoform-specific antibody and colloidal-gold secondary antibodies, an estimate of the amount of topo IIα in individual nuclear matrices. There are significant variations in topo IIα amounts between individual nuclear matrices due to the cell cycle distribution. The parental cell line contained eight to ten times more nuclear matrix–associated topo IIα than the resistant cell line matrices. Nuclear matrix–associated topo IIα from wild-type and resistant cell lines correlated well with the immunofluorescent staining of the enzyme in nuclei of intact cells. The amount of DNA associated with residual nuclear structures was five times greater in the resistant cell line. This quantity of DNA was not proportional to the quantity of topo IIα in the same matrix; in fact they were inversely related. In situ whole-mount nuclear matrix preparations were obtained from cells grown on grids and confirmed the results from labeling of isolated residual structures. J. Cell. Biochem. 67:112–130, 1997. © 1997 Wiley-Liss, Inc.     

DNase-hypersensitive sites in yeast artificial chromosomes containing human DNA

We have mapped the DNase I-hypersensitive sites (HSs) in Yeast Artificial Chromosomes (YACs) containing segments of human chromosomal DNA. One of the five HSs found in a YAC carrying the β-globin gene cluster has been localised in the region, termed HS2, that is DNase I hypersensitive in most human cells. We have also identified a class of HSs in YACs containing DNA from the q11.2 band of human chromosome 21, which are located close to, or within, segments of the chromosome that are sensitive to restriction enzymes recognizing CGCG tetranucleotides. Received: 18 June 1997 / Accepted: 10 August 1997     

A simple and accurate method for determination of microsatellite total allele content differences between DNA pools

DNA pooling is a potential tool for the efficient analysis of the large numbers of samples and DNA markers that are necessary for genome-wide association studies. A simple accurate method for measuring total allele differences in comparisons between two pools containing large numbers of DNA samples is presented. This method compares relative peak height differences between electrophoretograms for each allele of a microsatellite. The method was evaluated by the analysis of 11 microsatellite markers and DNA pooled sample sizes of 50, 100, and 200 individual DNA samples from the same number of different subjects. Pools were created from previously individually genotyped subjects and constructed so that the pool comparisons would provide real total allele differences varying from 0% to 55%. Calculated pool differences were then compared with the real total allele differences determined by individual genotyping results. Together over 200 comparisons demonstrated a correlation coefficient of 0.96, which compared favorably with other previous methods of analysis. This method could provide a rapid screen for total allele differences of greater than 10%, a threshold that should be applicable to detecting low relative risk genes in common diseases. Therefore, these studies suggest that DNA pooling could be a useful tool in association studies for the determination of candidate regions for a range of complex genetic diseases.     

DNA melting profiles from a matrix method

In this article we give a new method for the calculation of DNA melting profiles. Based on the matrix formulation of the DNA partition function, the method relies for its efficiency on the fact that the required matrices are very sparse, essentially reducing matrix multiplication to vector multiplication and thus making the computer time required to treat a DNA molecule containing N base pairs proportional to N(2). A key ingredient in the method is the result that multiplication by the inverse matrix can also be reduced to vector multiplication. The task of calculating the melting profile for the entire genome is further reduced by treating regions of the molecule between helix-plateaus, thus breaking the molecule up into independent parts that can each be treated individually. The method is easily modified to incorporate changes in the assignment of statistical weights to the different structural features of DNA. We illustrate the method using the genome of Haemophilus influenzae.     

Initiated complexes of RNA polymerase II are concentrated in the nuclear skeleton associated DNA

Several preparations of nuclear matrices containing varying amounts of DNA were obtained from mouse plasmocytoma P3-X63-Ag8.653 cells and tested for the presence of RNA polymerase II activity. It has been demonstrated that about 25% of RNA polymerase II activity detected in the original nuclei can be recovered in isolated nuclear matrices. Only DNA-bound RNA polymerase II was found in the isolated matrices, while both free and DNA-bound RNA polymerase II activities were detected in the original nuclei. RNA polymerase II activity found in the isolated matrices did not depend on the portion of DNA recovered in the nuclear matrices in a large interval between 91 and 1.5% of DNA content in the original nuclei. The conclusion has been drawn that initiated RNA polymerase II molecules are non-randomly distributed along DNA loops. They are concentrated near the points of DNA attachment to the nuclear skeleton.     

Endemicity biases nestedness metrics: a demonstration, explanation and solution

Nestedness is frequently investigated to understand complex patterns of species occurrences. Temperature (T) is commonly used for comparisons of nestedness of different-sized datasets. However, the assumptions made for the standardization of this metric have not been fully explored, particularly the effects of endemicity. Here we show that T incorrectly indicates an increase in nestedness with the addition of non-nested endemics to matrices that are not perfectly nested – a consequence of standardizing matrix size by the product of species and sites. This problem is common both to test matrices and to real matrices that are typically subjected to nestedness analyses. The latter are often characterized by substantial numbers of endemics and by variation in the numbers of endemics in different taxa. Standardizing by occupancy resolves this problem, which is demonstrated using a derivative of discrepancy, d1. A small modification to T, such that it standardizes matrices by occupancy, would resolve the current problems with this nestedness metric.     

Genetic structure of the indigenous populations of Siberia

This study explores the genetic structure of Siberian indigenous populations on the basis of standard blood group and protein markers and DNA variable number of tandem repeats (VNTR) variation. Four analytical methods were utilized in this study: Harpending and Jenkin's R-matrix; Harpending and Ward's method of correlating genetic heterozygosity (H) to the distance from the centroid of the gene frequency array (r_ii); spatial autocorrelation, and Mantel tests. Because of the underlying assumptions of the various methods, the numbers of populations used in the analyses varied from 15 to 62. Since spatial autocorrelation is based upon separate correlations between alleles, a larger number of standard blood markers and populations were used. Fewest Siberian populations have been sampled for VNTRs, thus, only a limited comparison was possible. The four analytical procedures employed in this study yielded complementary results suggestive of the effects of unique historical events, evolutionary forces, and geography on the distribution of alleles in Siberian indigenous populations. The principal components analysis of the R-matrix demonstrated the presence of populational clusters that reflect their phylogenetic relationship. Mantel comparisons of matrices indicate that an intimate relationship exists between geography, languages, and genetics of Siberian populations. Spatial autocorrelation patterns reflect the isolation-by-distance model of Malecot and the possible effects of long-distance migration. Am J Phys Anthropol 104:177–192, 1997. © 1997 Wiley-Liss, Inc.     

Microbial colonization of polyurethane foam matrices in horizontal-flow anaerobic immobilized-sludge reactor

This paper presents the anaerobic biomass characterization and the bacterial framework inside polyurethane foam matrices taken from a horizontal-flow anaerobic immobilized-sludge (HAIS) reactor treating a glucose-based substrate. Ultrastructure polyurethane foam analyses carried out using scanning electron microscopy (SEM) in samples treated with hexamethyldisylazane showed three different patterns of biomass retention inside the polyurethane foam matrices: micro-granules ranging from 270 μm to 470 μm were entrapped in the porous medium thin multi-cellular films were attached to the inner surface, and individual cells adhered to the support. The use of SEM and epifluorescence microscopy permitted inferences to be made on the bacteriological composition of the immobilized sludge formed by different morphotypes (rods, cocci and filaments) and on the ecological significance of their framework inside the matrices. Polyurethane matrices were found to offer excellent conditions for anaerobic growth and retention, favoring the flux of substrate and products. Such outstanding characteristics were confirmed by the short start-up period observed during the operation of the HAIS reactor. Received: 4 April 1997 / Received revision: 28 May 1997 / Accepted: 1 June 1997     

Reduction of DNA primase activity in aging but still proliferating cells

The basis of the well-known decline in cell proliferation with increasing passage number of human diploid fibroblast-like cell cultures is not known. It has been found that DNA synthesis was deficient in the remaining but still proliferating cells, but when appropriate corrections reflecting the remaining dividing cells were made, the amount of DNA polymerase alpha bound to nuclear matrices was normal [Collins and Chu: Journal of Cellular Physiology 124:165-173, 1985]. In the present study, the declining percentages of S-phase and dividing cells were determined to provide better estimates of functional culture age than passage number. The amounts of DNA polymerase alpha and DNA primase activity were determined in cell lysates, permeabilized cells, and bound to nucleoids, which are residual nuclear structures similar to nuclear matrices except that no DNase-digestion step is employed. As expected, IMR 90 DNA synthesis declined with age, even after corrections for the declining numbers of proliferating cells. DNA polymerase alpha and DNA primase activity in cell lysates, permeabilized cells, and bound to nucleoids declined with increasing age. However, after appropriate corrections for the declining fraction of proliferating cells, the only activity that declined was that of DNA primase bound to nucleoids. Thus, a decrease in the binding of DNA primase to the nuclear site of DNA synthesis may account for the decreased DNA synthesis in aging but still proliferating cells.     

A Likelihood-Based Trait-Model-Free Approach for Linkage Detection of Binary Trait

Summary .  Trait-model-free (or "allele-sharing") approach to linkage analysis is a popular tool in genetic mapping of complex traits, because of the absence of explicit assumptions about the underlying mode of inheritance of the trait. The likelihood framework introduced by Kong and Cox (1997,  American Journal of Human Genetics   61, 1179–1188) allows calculation of accurate p-values and LOD scores to test for linkage between a genomic region and a trait. Their method relies on the specification of a model for the trait-dependent segregation of marker alleles at a genomic region linked to the trait. Here we propose a new such model that is motivated by the desire to extract as much information as possible from extended pedigrees containing data from individuals related over several generations. However, our model is also applicable to smaller pedigrees, and has some attractive features compared with existing models ( Kong and Cox, 1997 ), including the fact that it incorporates information on both affected and unaffected individuals. We illustrate the proposed model on simulated and real data, and compare its performance with the existing approach ( Kong and Cox, 1997 ). The proposed approach is implemented in the program lm_ibdtests within the framework of MORGAN 2.8 ( http://www.stat.washington.edu/thompson/Genepi/MORGAN/Morgan.shtml ).     

Hamster chromosomes containing amplified human α-satellite DNA show delayed sister chromatid separation in the absence of de novo kinetochore formation

The centromeres of human chromosomes contain large amounts of the tandemly repeated α-satellite DNA family. Previous studies have shown that integration of α-satellite DNA into ectopic locations in mammalian chromosomes can result in the de novo formation of several features of centromeric function. Here we further examine the possible centromeric properties of α-satellite DNA by introducing it into hamster chromosomes. A large amplified region of ectopic α-satellite DNA was shown to direct binding of anticentromere antibodies (ACAs) and centromere protein B (CENP-B). The chromosome containing these ectopic arrays showed a high frequency of formation of anaphase bridges. Owing to the favourable morphology of these chromosomes, we were able to determine that this bridging was due to delayed sister chromatid disjunction at the location of the ectopic α-satellite, and not due to de novo formation of a fully functional kinetochore. A separate hamster cell line containing large tandemly repeated amplicons including the DHFR gene also displayed similar behaviour during anaphase. These results may support a role for α-satellite DNA in sister chromatid cohesion at centromeres. However, other repetitive DNA in favourable configurations appears to be capable of mimicking this behaviour during anaphase. Received: 31 December 1996; in revised form: 14 February 1997 / Accepted: 24 February 1997     

Calculating the intrinsic growth rate: comparison of definition and model

It was shown that well known equation r = ln[N(t2)/N(t1)]/(t2 - t1) is the definition of the average value of intrinsic growth rate of population r within any given interval of time t2-t1 and changing arbitrarity its numbers N(t). The common opinion considering the equation as suitable only for exponentially growing population was found to be incorrect. The fundamentally different approach is based on the calculation of r within the framework of demographic model, realized as Euler - Lotka equation or population projection matrices. However this model requires simultaneous realization of several assumptions improbable for natural populations: exponential change in population size, stable age structure and maintaining constant age-dependent birth and death rates. The calculation of r by definition requires the data on the dynamics of population numbers, whereas calculation on the basis of the model requires the demographic tables of birth and death rate, but not the population numbers. With the example of American ginseng it was shown that evalution of r by definition and model approaches could produce opposite results.     

DNA replication asynchrony between the paternal and maternal alleles of imprinted genes does not straddle the R/G transition

Imprinted autosomal loci apparently reside in very large chromosomal domains that exhibit asynchrony in replication of homologous alleles during the DNA synthesis phase. Replication asynchrony can be cytogenetically visualized by a replication-banding discordance between homologous bands of a given pair of chromosomal homologs. The replication time of a chromosomal band at high resolution can be determined by blocking DNA synthesis at the R/G-band transition and using replication banding. The R/G transition reflects the transition from early (R-) to late (G- and C-) band DNA replication. We studied discordance between two groups of homologous chromosomal bands: (a) four bands, 6q26–27, 11p13, 11p15.5 and 15q11.2–12, each containing at least one imprinted gene; and (b) nine bands containing no known imprinted genes. Fifty pairs of chromosomes were analyzed at high resolution after R/G transition blocking and late 5-bromo-2′-deoxyuridine incorporation. The rate of discordance was the same for bands containing imprinted genes and for control bands. Both homologous bands of a pair replicate either before or after the R/G transition and do not straddle the R/G transition. Repression associated with imprinting does not appear to involve late replication at the band level of resolution. Tissue-specific inactivation is associated with DNA methylation and late replication, whereas allele-specific inactivation is associated with DNA methylation but not with delayed or late replication. Received: 7 May 1996; in revised form: 27 January 1997 / Accepted: 31 July 1997     

RecA-mediated joint molecule formation between O-methylated RNA/DNA hairpins and single-stranded targets

Chimeric oligonucleotides consisting of one DNA strand paired with an O-methylated RNA strand interrupted by six DNA residues have been used in gene targeting experiments. Here we demonstrate that these hairpins can form a heteroduplex (or joint molecule) with single-stranded DNA targets in a reaction mediated by the E. coli RecA protein. One end of the double hairpin may unwind to form a 14-base-RecA filament which initiates the reaction. Chimeric oligonucleotides containing only O-methylated RNA residues on one strand or truncated hairpins lacking this 14-base segment did not participate in RecA-driven heteroduplex formation under these reaction conditions. The results presented here represent a first step in studying one facet of a strategy which uses O-methylated RNA residues as participants in homologous pairing events. Received: 2 June 1997 / Accepted: 29 September 1997     

Tyrosination-detyrosination of tubulin and microtubules during the development of chick erythrocytes

The protein composition of nuclear matrices containing different amount of DNA was examined. It was found that, in matrices containing 2% to 80% of total DNA, the quantity of DNA-bound proteins remains relatively constant varying from 10% to 15% of total nuclear proteins. Electrophoretic patterns do not differ substantially, but autoradiograms with in vitro ¹²⁵I labelled proteins show quantitative variations in the actin content. Application of radioimmunoassay (RIA) enabled to determine the exact content of actin in GAT nuclei and nuclear matrices – 5 g/ml in nuclei, of which 50% are bound to DNA and 3001o being a component of the protein part of the nuclear matrix. These results are supported by electron microscopic data, where immunogold technique was performed on thin sections and spread material. The applied methods suggest that part of the nuclear actin is tightly bound (resistant to 2 M NaCI) to DNA and represents a component of the internal nuclear matrix.     

Detection of adeno- and lentiviral (HIV1) contaminations on laboratory surfaces as a tool for the surveillance of biosafety standards

Aims: As a biosafety laboratory, we survey the handling of adenovirus type 5 (Ad5) and HIV1‐derived lentivirus in contained‐use facilities in Switzerland to identify insufficiencies of the safety precautions taken by the laboratories. Methods and Results: In the past 9 years, we took 430 swab samples from various types of surfaces in research laboratories. Samples were examined for Ad5 contaminations by real‐time PCR and infectivity assay or for the presence of lentivirus (HIV1) nucleic acids by real‐time (RT) PCR. Samples collected from centrifuges did not only contain Ad5 DNA more frequently but also exhibited higher numbers of Ad5 and lentiviral (HIV1) DNA copies than swabs from any other area of sampling. Five of ten samples containing infectious Ad5 particles or lentivirus (HIV1) RNA were found in samples taken from centrifuges. Ad5 contamination rates were higher in the tube holder and lower on the inner wall of the rotor chamber in centrifuges that were fitted with aerosol tight covers compared to centrifuges without covers. Conclusions: Our results allowed the comparison of hygiene standards of different laboratories and lead to the identification of centrifuges as hotspots for contaminations. Significance and Impact of the Study: Based on our results, we propose to use the collected data as a tool for rating future swab results. Furthermore, the amount of Ad5 and HIV1‐derived lentivirus DNA could serve as an indicator of the level of good laboratory practice in contained‐use laboratories handling these viral vectors.     

DNA delivery from polymer matrices for tissue engineering.

We have proposed engineering tissues by the incorporation and sustained release of plasmids encoding tissue-inductive proteins from polymer matrices. Matrices of poly(lactide-co-glycolide) (PLG) were loaded with plasmid, which was subsequently released over a period ranging from days to a month in vitro. Sustained delivery of plasmid DNA from matrices led to the transfection of large numbers of cells. Furthermore, in vivo delivery of a plasmid encoding platelet-derived growth factor enhanced matrix deposition and blood vessel formation in the developing tissue. This contrasts with direct injection of the plasmid, which did not significantly affect tissue formation. This method of DNA delivery may find utility in tissue engineering and gene therapy applications.     

Inhibition of shoot induction by 5-azacytidine and 5-aza-2′-deoxycytidine in Petunia involves DNA hypomethylation

Shoot bud regeneration from Petunia leaf disks was inhibited when they were cultured with the demethylating agents, 5-azacytidine (AzaC) and 5-aza-2′-deoxycytidine (AzadC), in shoot induction (SI) medium. Explants induced shoot primordia if they were transferred after 1 week from the medium containing the drugs to medium without drugs. The fresh weight of leaf disks cultured on SI medium for 2 weeks in the presence of the drugs was 60–80% lower when compared to control shoot-forming cultures. Internode length was reduced when shoots were transferred to phytohormone-free Murashige and Skoog medium containing the drugs. However, no other morphological abnormalities were seen in these shoots, even at 20 μm AzaC or 5 μm AzadC. Coupled restriction enzyme digestion (with HpaII and MspI) and random amplification of genomic DNA was performed to detect the level of methylation of CCGG sites in the DNA of the explants exposed to AzaC and AzadC. Over 15 amplified bands were detectable in the control. Five of these bands were absent in the amplified products when digested DNA from the drug-treated explants was used as the template, showing that hypomethylation of DNA had occurred. This suggests that inhibition of shoot bud formation in the presence of the drugs AzaC and AzadC may be due to the altered methylation status. Received: 7 January 1997 / Revision received: 17 February 1997 / Accepted: 1 March 1997     

Biolistic transfer and expression of a uidA reporter gene in different tissues of Allium sativum L.

The biolistic particle delivery system was used to transfer the uidA gene into different garlic tissues, including regenerable calli. These tissues showed a high endogenous nuclease activity preventing exogenous DNA expression. After submerging the tissues for 24 h in a 2 mm solution of the nuclease inhibitor aurintricarboxylic acid, transient expression of the uidA gene was detected. Furthermore, vacuum infiltration of aurintricarboxylic acid into the garlic tissues significantly increased the percentage of explants showing transient expression. Among four plasmids tested, pDE4 containing the CaMV35S-uidA construct produced the best results. Received: 16 May 1997 / Revision received: 26 August 1997 / Accepted: 25 September 1997